The present invention, in some embodiments thereof, relates to methods of generating antibodies to metalloenzymes, more particularly, but not exclusively, to MMP-7 and anthrax lethal factor (ALF).
The matrix metalloproteins (MMPs) are key enzymes participating in remodeling of the extracellular matrix (ECM). These enzymes are capable of destroying a variety of connective tissue components of articular cartilage or basement membranes.
The human MMP gene family consists of at least 28 structurally related proteins, which share a similar overall spherical topology. Each MMP is secreted as an inactive, latent pro-enzyme. The catalytic zinc domain is composed of about 180 amino acids wherein the highly conserved sequence HE-GH-LGL-H provides the three histidine (i.e., H) residues which bind to the metal Zn(2+) ion. The forth-binding site of the catalytic zinc ion in the pro-enzyme is bound to a cystein residue (Morgunova et al., 1999), which upon enzyme activation dissociates from the active site (Van Wart and Birkedal-Hansen, 1990). As a result, the forth-binding site in the activated MMPs is taken up by a water molecule, which is also hydrogen-bonded to a conserved glutamic residue. This process facilitates the hydrolysis of a peptide bond of the target substrate with the activated water molecule.
MMP-7 (also referred to in the literature as “matrilysin”) is expressed in epithelial cells of normal and diseased tissues, and is capable of digesting a large series of proteins located in the extracellular matrix. These include collagen IV and X, gelatin, casein, laminin, aggrecan, entactin, elastin and versican. MMP-7 appears to play a role in the activation of other proteinases such as plasminogen, MMP-1, MMP-2, and MMP9. In addition to its role in connective tissue remodeling, MMP-7 has been shown to be expressed in some malignant tumors and may play an important role in tumor invasion and metastasis. Structurally, MMP-7 is the smallest of the MMPs and consists of two domains: a pro-domain that is cleaved upon activation and a catalytic domain containing the zinc-binding site. Several publications have shown that the MMP proteins, and in particular MMP-7, are implicated in ovarian and other cancers such as renal cell carcinoma. It has also been suggested to be a candidate biomarker for ovarian cancer—see, for example, Wang et al. (2005) Int. J. Cancer 114(1):19-31; Wang et al. (2006) Int. J. Cancer 118(4):879-88; Maurel et al. (2007) Int. J. Cancer 121(5): 1066-1071; and Sarkissian et al. (2008) Clin. Chem. 54(3): 574-581.
Anthrax is a highly lethal infectious disease caused by the spore-forming bacterium Bacillus anthracis. The deliberate distribution of anthrax spores through US mail system in 2001 resulted in 5 deaths among the 11 individuals who contracted inhalational anthrax, which highlight the great threat posed by the potential use of anthrax in terrorism and warfare. The lethality of inhalational anthrax is primarily due to the action of anthrax toxins. Bacterium produces three toxin components; they are protective antigen (PA), lethal factor (LF), and edema factor (EF). PA together with LF forms lethal toxin (LT) and PA together with EF forms edema toxin (ET). PA functions as a vehicle to mediate the cellular uptake of the LF and EF. LF is a metalloenzyme that cleaves mitogen-activated protein kinase kinases (MEKs) and can replicate symptoms of anthrax disease when injected in animals with PA. EF is a calcium-calmodulin-dependent adenylate cyclase with a range of toxic effects in the host. These toxins are the dominant virulence factors for anthrax disease.
Currently there are no approved therapies for anthrax disease except antibiotics. Treatment with antibiotics, though, has considerable limitations. Exposure to the bacterium followed by bacterial division leads to the production of large quantities of the anthrax toxins. Thus, unless exposure is diagnosed early enough for vigorous antibiotic treatment, patients will succumb to disease even after the killing of all bacteria. The current vaccine approved by US Food and Drug Administration is also not effective in protecting newly infected individuals, as it requires repeated administration and at least 4 weeks for development of protective titers.
International Patent Application WO2004/087042 and U.S. Pat. No. 8,324,355 teaches the generation of antibodies targeted at the catalytic zinc ion and the enzyme surface of MMPs which are naturally expressed in animals.
U.S. Patent Application No. 20100119520 teaches monoclonal antibodies directed against anthrax lethal factor.
U.S. Patent Application No. 20100227335 teaches monoclonal antibodies directed against MMP-7.